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First published online September 7, 2006
doi: 10.1242/10.1242/jcs.03151

Journal of Cell Science 119, 3776-3787 (2006)
Published by The Company of Biologists 2006
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Syntaxin 1A promotes the endocytic sorting of EAAC1 leading to inhibition of glutamate transport

Yong-Xin Yu1,2, Li Shen1, Peng Xia1, Ya-Wei Tang1, Lan Bao1,* and Gang Pei1,*

1 Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences
2 Graduate School of Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, People's Republic of China


Figure 1
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  		Fig. 1. Syntaxin 1A facilitates EAAC1 endocytic sorting. (A) Representative immunoblot and quantitative analysis showing that in C6 glioma cells transfected with syntaxin 1A (Syn1A), the expression of endogenous EAAC1 is decreased in both total lysate and cell surface fractions. Actin served as an internal control for protein loading. Data are plotted as a percentage of EAAC1 in the cells transfected with ß-gal within each fraction. **P<0.01 compared with cells transfected with ß-gal (n=3). (B) Representative immunoblot and quantitative analysis show that the delivery efficiency of EAAC1 is not altered in Syn1A-transfected C6 glioma cells. The data are plotted as a percentage of the increased cell surface fraction versus the intracellular EAAC1 pool available for surface delivery (n=3). (C) Representative immunoblot and quantitative analysis of internalized EAAC1 show that the internalization of EAAC1 is potentiated in Syn1A-transfected C6 glioma cells (upper panel), despite syntaxin 1A decreasing the overall EAAC1 surface pool available for internalization (lower panel). The band intensities for the internalized EAAC1 are plotted as a percentage of the total surface EAAC1 available for internalization within each transfection group. **P<0.01 compared with the cells transfected with ß-gal at the same time point (n=3).

 

Figure 2
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  		Fig. 2. Syntaxin 1A specifically inhibits EAAC1-mediated glutamate transport. In C6 glioma cells, the transfection of Syn1A and specific Syn1A siRNA (Syn1A siRNA) are indicated (A-D). In HEK293 cells, Syn1A are co-transfected pairwise with EAAC1, EAAT4 and GLT1; EAAC1 is co-transfected pairwise with syntaxin 4, SNAP-25 and VAMP-2 as indicated (E-G). (A) Syntaxin 1A significantly inhibits EAAC1 transport activity in C6 glioma cells. **P<0.01 compared with the cells transfected with ß-gal (n=4). (B) Kinetics analysis of glutamate transport in C6 glioma cells transfected with Syn1A. (Left panel) Saturation analysis of glutamate transport shows that Syn1A decreases transport maximal velocity compared with the cells transfected with ß-gal, whereas Km is nearly identical. (Right panel) Eadie-Hofstee transformations of these data (n=4). (C) Specific siRNA of Syn1A increases EAAC1 transport activity in C6 glioma cells. **P<0.01 compared with the cells transfected with non-specific siRNA (NS siRNA) (n=5). (D) Kinetics analysis of glutamate transport in C6 glioma cells transfected with Syn1A siRNA. (Left panel) Saturation analysis shows that Syn1A siRNA increases EAAC1 transport maximal velocity compared with the cells transfected with NS siRNA, whereas Km is nearly identical. (Right panel) Eadie-Hofstee transformations of these data (n=5). (E) EAAC1 transport activity is decreased progressively with increasing amounts of Syn1A plasmid. The HEK293 cells are triple-transfected with EAAC1, Syn1A, and ß-gal expression plasmids as indicated. **P<0.01 compared with control (n=3). (F) Syn1A has no effect on the transport activity of EAAT4 or GLT1 (n=3). (G) The transport activity of EAAC1 is not regulated by syntaxin 4, SNAP-25 or VAMP-2 (n=3).

 

Figure 3
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  		Fig. 3. Regulation of the clathrin-mediated EAAC1 internalization by syntaxin 1A. (A) Representative immunoblot and quantitative analysis showing that hypertonic medium containing 0.45 M sucrose increases EAAC1 surface expression and glutamate transport activity. **P<0.01 compared with the control (n=3). (B) The C6 glioma cells are transfected with ß-gal, wild-type dynamin (Dyn), or K44A dynamin (K44A) constructs. Representative immunoblot showing that the EAAC1 surface expression is decreased by transfection of dynamin but not by K44A dynamin. (C) Representative immunoblot of surface biotinylated-EAAC1 shows that Syn1A siRNA significantly rescues the dynamin-induced decrease in EAAC1 surface expression.

 

Figure 4
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  		Fig. 4. Syntaxin 1A H3 and transmembrane domains mediate the association with EAAC1. (A) Coimmunoprecipitation (IP) and immunoblotting (IB) show that in HEK293 cells co-transfected with Syn1A and EAAC1, Syn1A is found in EAAC1-antibody-precipitated proteins and vice versa. The lysate and the immunoprecipitated proteins are analyzed on the same gel. Data represent three independent experiments. (B) Coimmunoprecipitation and immunoblotting show that in the mouse hippocampal extracts and C6 glioma cells, Syn1A is found in the EAAC1 antibody-precipitated proteins and indicate that Syn1A interacts with EAAC1 in vivo. The lysate and the immunoprecipitated proteins are analyzed on the same gel. The data represent three independent experiments. (C) Representative immunoblot showing that a high-molecular-mass band at ~100 kDa is detected by both EAAC1 (left panel) and Syn1A (right panel) antibodies in EAAC1 antibody-precipitated proteins from the C6 glioma cells cross-linked with BS3. Data represent three independent experiments. (D) Diagram illustrates wild-type and mutants of Syn1A including H3 and transmembrane domains (Syn1A H3-TMD), H3 domain (Syn1A H3), and H3 domain deletion (Syn1A {Delta}H3), and summarizes the results of the interaction and regulation of EAAC1 by these molecules tagged with HA in HEK293 cells. (E) In HEK293 cells co-transfected with EAAC1 and HA-Syn1A H3-TMD, HA-immunoreactive band is found at the position of Syn1A H3-TMD in EAAC1-antibody-precipitated protein. The lysate and the immunoprecipitated proteins are analyzed on the same gel. The data represent three independent experiments. (F) [3H]glutamate uptake shows that Syn1A H3-TMD has a similar inhibition on EAAC1 transport activity as Syn1A, whereas Syn1A {Delta}H3 and Syn1A H3 have no effect on EAAC1 transport activity. **P<0.01 compared with the cells co-transfected with ß-gal (n=3). Consistently, representative immunoblot shows that the cell surface expression of EAAC1 is decreased by co-transfection of Syn1A H3-TMD, whereas Syn1A {Delta}H3 and Syn1A H3 have no effect on EAAC1 surface expression.

 

Figure 5
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  		Fig. 5. Essential role of syntaxin 1A in KA-promoted EAAC1 internalization. (A) Representative immunoblot and quantitative analysis showing that stimulation with 10 µM KA causes a decrease in EAAC1 surface expression in C6 glioma cells, which is rescued by transfection of K44A dynamin. *P<0.05, **P<0.01 compared with the cells without KA stimulation (n=3). (B) Stimulation with KA for 30 minutes inhibits the EAAC1-mediated glutamate transport in C6 glioma cells (upper panel), which is correlated with a decrease in the surface expression of EAAC1 (lower panel). **P<0.01 versus the cells without KA stimulation (n=3). (C) Representative immunoblot and quantitative analysis of internalized EAAC1 upon KA stimulation for various time show constitutive internalization of EAAC1, and that KA stimulation significantly promotes EAAC1 internalization in C6 glioma cells. **P<0.01 compared with cells without KA stimulation at identical time points (n=4). (D) Representative immunoblot of surface EAAC1 expression upon KA stimulation for various time showing that Syn1A siRNA rescues KA-induced reduction in EAAC1 surface expression in C6 glioma cells. (E) Representative immunoblot of internalized EAAC1 upon KA stimulation for various time showing that Syn1A siRNA abolishes the KA-promoted EAAC1 internalization in C6 glioma cells.

 

Figure 6
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  		Fig. 6. KA stimulation promotes the degradation of internalized EAAC1 in the lysosome. (A,B) Double-immunofluorescence labeling showing that internalized EAAC1 is colocalized with the early endosomal marker EEA1 (A) and the lysosomal probe LysoTracker Red DND-99 (B) in C6 glioma cells upon a 30-minute stimulation with 10 µM KA. Bars, 10 µm. Scatterplot graphs of the individual pixels from the paired images and the Pearson's correlations calculated by Image-Pro-Plus software, suggesting that the colocalization of EAAC1 with EEA1 or Lysotracker Red DND-99 is significantly enhanced after KA stimulation. (C) Representative immunoblot and quantitative analysis show that incubation with 10 µM KA for 6 hours leads to the degradation of the internalized EAAC1, which is blocked by the lysosomal degradation inhibitor leupeptin (100 µM) or the compartment acidification blockers NH4Cl (50 mM) and chloroquine (200 µM). **P<0.01 compared with the cells without KA stimulation (n=4). (D) Representative immunoblot and quantitative analysis showing that upon 10 µM KA stimulation the degradation of the internalized EAAC1 increases in a time-dependent manner. Additionally, Syn1A siRNA blocks the KA-promoted EAAC1 degradation. *P<0.05, **P<0.01 compared with the cells transfected with NS siRNA (n=4).

 

Figure 7
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  		Fig. 7. KA stimulates EAAC1 internalization in primary cultured neurons. (A) Representative immunoblot and quantitative analysis of internalized EAAC1 upon KA stimulation in the primary cultured hippocampal neurons for various time showing that there is a constitutive internalization of EAAC1 that is significantly promoted by KA stimulation. *P<0.05, **P<0.01 compared with the cells without KA stimulation at identical time points (n=3). (B) Representative immunoblot and quantitative analysis show that stimulation with 10 µM KA decreases EAAC1 surface expression in the primary cultured hippocampal neurons. *P<0.05, **P<0.01 compared with the cells without KA stimulation (n=3).

 

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© The Company of Biologists Ltd 2006